Biomed Res Int. 2014;2014:502542.

Endocannabinoid Receptors Gene Expression in Morbidly Obese Women with Nonalcoholic Fatty Liver Disease

Teresa Auguet1,2, Alba Berlanga1, Esther Guiu-Jurado1, Ximena Terra1, Salomé Martinez3, Carmen Aguilar1, Elisa Filiu2, Ajla Alibalic2, Fàtima Sabench4, Mercé Hernández4, Daniel Del Castillo4, and Cristóbal Richart1,2

1 Grup GEMMAIR (AGAUR) and Grup de Recerca en Medicina Aplicada, Departament de Medicina i Cirurgia, IISPV, Hospital Universitari Joan XXIII, Universitat Rovira i Virgili (URV), Mallafr´e Guasch 4, Catalonia, 43007 Tarragona, Spain

2 Servei Medicina Interna, Department of Internal Medicine, Hospital Universitari de Tarragona Joan XXIII, Universitat Rovira i Virgili, Mallafr´e Guasch 4, Catalonia, 43007 Tarragona, Spain

3 Servei Anatomia Patol`ogica, Hospital Universitari Joan XXIII Tarragona, Mallafré Guasch 4, Catalonia, 43007 Tarragona, Spain

4 Servei de Cirurgia, Hospital Sant Joan de Reus, Avenida del Dr. Josep Laporte 2, Catalonia, Tarragona, 43204 Reus, Spain

 

ABSTRACT

Background. Recent reports suggest a role for the endocannabinoid system in the pathology of nonalcoholic fatty liver disease (NAFLD). The aim of this study was to investigate the relationship between liver expression of cannabinoid (CB) receptor subtypes, CB1 and CB2, in morbidly obese (MO) women with different histological stages of NAFLD.

Methods. We analysed hepatic CB1 and CB2 mRNA expression, and the expression of genes involved in lipidmetabolism in 72MO women, subclassified by liver histology into MO with normal liver (NL, n = 16), simple steatosis (SS, n = 28), and nonalcoholic steatohepatitis (NASH, n = 28) by enzyme-linked immunosorbent assay and RT-PCR.

Results. We found that CB1mRNAexpressionwas significantly higher in NASH compared with SS and correlated negatively with PPARalpha. Regarding CB2, CB2 mRNA expression correlated positively with ACC1, PPARalpha, IL6, TNFalpha, resistin, and adiponectin.

Conclusions. The increased expression of CB1 in NASH and the negative correlation with PPARalpha suggest a deleterious role of CB1 in NAFLD. Regarding CB2, its positive correlation with the anti-inflammatory molecule adiponectin and, paradoxically, with inflammatory genes suggests that this receptor has a dual role. Taken together, our results suggest that endocannabinoid receptors might be involved in the pathogenesis of NAFLD, a finding which justifies further study.

PMID: 24864249

 

SUPPLEMENT:

Obesity, as part of the metabolic syndrome, is one of the major risk factors in the development of fatty liver 1,2. Nonalcoholic fatty liver disease (NAFLD) has become the most common liver disorder in developed countries, affecting over one-third of the population 3,4. NAFLD has frequently been associated with obesity, type 2 diabetes mellitus, hyperlipidemia, and insulin resistance 5. The spectrum of the disease ranges from simple steatosis to steatohepatitis, a condition that associates steatosis, liver inflammation, hepatocellular injury, and activation of fibrogenic pathways with a 10–20% risk of developing cirrhosis within 10 to 20 years 6. The pathogenic transition from steatosis (SS) to nonalcoholic steatohepatitis (NASH) is not completely understood and appears multifactorial. Recent studies have revealed a role of lipotoxic fatty acid metabolites originating from the adipose tissue or from de novo lipogenesis in the development of hepatocellular injury 7. Increasing evidence suggests that a fatty liver is more vulnerable to factors that lead to inflammation and fibrosis 8,9. Different studies confirm that de novo lipogenesis might be upregulated in NAFLD. 10–12Recent reports have shown that endogenous cannabinoids (EC) are related to fatty liver metabolism 13,14 although the molecular mechanism by which EC modulates the metabolism within hepatocytes is still not clear.

Endogenous cannabinoids are lipid mediators that produce similar effects to those of marijuana by acting on membrane-bound receptors and regulating appetite behavior 15. They exert their effects through two different cannabinoid receptors, CB1 and CB2, which are mainly localized in the brain, but are also present in small amounts in liver and other peripheral tissue (CB1) and in immune and haematopoietic cells (CB2) 16,17. Under physiological conditions, the EC system is silent, since CB1 and CB2 receptors are faintly expressed. In contrast, induction of CB receptors and/or increased lev¬els of ECs are common features of liver injuries of diverse origins 18. Both receptors have been implicated in the devel¬opment of liver fibrosis secondary to various etiologies.
CB1-mediated EC tone is enhanced in experimental diet-induced or genetic models of NAFLD, and is characterized by upregulation of adipose tissue and hepatocyte CB1 recep-tors, and by increased liver synthesis of anandamide. The pathogenic role of CB1 receptors in NAFLD is supported by the resistance to steatosis of obese mice bearing a global or hepatocyte-specific CB1 deletion, or of rodents administered rimonabant or AM6545, a CB1 antagonist 19–21. Studies with cultured hepatocytes and liver slices further indicate that the steatogenic properties of CB1 arise from altered hepatic lipid metabolism, consisting of a combination of hepatocyte activation of SREBP1c-mediated lipogenesis, reduction of FA oxidation via inhibition of AMP kinase, and decreased release of TG-rich VLDL 19,20,22. In addition, the adipose tissue may largely contribute to the steatogenic process via CB1-induced release of FFAs by adipocytes 23(Figure 1). These findings indicate that CB1 receptors mediate metabolic steatogenesis in the liver by central and peripheral effects. Regarding CB2, results of recent studies have suggested that this receptor could be a promising anti-inflammatory and antifibrogenic target 24,25, although clinical development of its agonists is still awaited.

Based on that data, we wished to further investigate the associations of CB1 and CB2 with NAFLD. We first analysed hepatic CB1 and CB2 mRNA expression in liver of morbidly obese (MO) women with or without NAFLD. When we subclassified that cohort into normal liver (NL), SS, and NASH, we observed that CB1 mRNA expression was significantly higher in NASH compared with SS (SS: 0.09 ± 0.07; NASH: 0.14 ±0.03; p < 0.010) (Figure 2(a)). However, CB2 gene expression was similar among the three groups (Figure 2(b)). Then, we studied the relationship between liver CB1 and CB2 mRNA expression and liver mRNA expression of key genes involved in lipid metabolism: genes involved in de novo synthesis of fatty acids (ChREBP, SREBP1c, LxRalpha, FxR, ACC1, and FAS), fatty acid oxidation (PPARalpha), uptake and transport (PPARalpha, CD36, and FABP4), and inflammatory related genes (PPARalpha, IL6, TNFalpha, and CRP). We found a negative correlation between CB1 and PPARalpha gene expression. We also found that CB2 mRNA expression correlated positively with ACC1 and PPARalpha mRNA expression (Table 1). Regarding inflammation and adipokines, we did not find any correlation between CB1 gene expression and inflammatory genes expression, nor with adipokines expression. However, we found positive correlations between CB2 and IL6, TNFalpha, resistin, and adiponectin gene expression (Table 1). In addition, we performed a stepwise multiple linear regression analysis, which included age, BMI, triglycerides, PPARalpha, and the presence of NASH as independent variables, and CB1 expression as a dependent variable. The results indicated that NASH and PPARalpha (inverse) were the only variables associated with CB1 expression (R2= 0.255, P=0.002; R2= 0.129, P= 0.014, resp.).

The importance of this study is two-fold. First, the study cohort of morbidly obese women has made it possible to establish clear relationships between NASH and CB1 liver expression without the interference of such confounding factors as gender or age. However, it cannot be extrapolated to other obesity groups, to men, or to normal-weight subjects. The increased expression of CB1 in NASH and the negative correlation with PPARalpha suggest a deleterious role of CB1 in NAFLD. Second, regarding CB2, its positive correlation with the anti-inflammatory molecule adiponectin and, paradoxically, with inflammatory genes suggests that this receptor may have a dual role.

In conclusion, the study shows that endocannabinoid receptors might be involved in the physiopathological processes of NAFLD and justify the need for further study.

 

References:

1. Harrison, S. A. & Diehl, A. M. Fat and the liver–a molecular overview. Semin. Gastrointest. Dis. 13, 3–16 (2002).
2. Clark, J. M., Brancati, F. L. & Diehl, A. M. Nonalcoholic fatty liver disease. Gastroenterology 122, 1649–1657 (2002).
3. Angulo, P. Nonalcoholic Fatty Liver Disease. N. Engl. J. Med. 346, 1221–1231 (2002).
4. Vernon, G., Baranova, A. & Younossi, Z. M. Systematic review: the epidemiology and natural history of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis in adults. Aliment. Pharmacol. Ther. 34, 274–285 (2011).
5. Marchesini, G. et al. Nonalcoholic Fatty Liver, Steatohepatitis, and the Metabolic Syndrome. Hepatology 37, 917–923 (2003).
6. Ong, J. P. & Younossi, Z. M. Epidemiology and natural history of NAFLD and NASH. Clin. Liver Dis. 11, 1–16, vii (2007).
7. Tilg, H. & Moschen, A. R. Evolution of inflammation in nonalcoholic fatty liver disease: the multiple parallel hits hypothesis. Hepatology 52, 1836–1846 (2010).
8. Postic, C. & Girard, J. Contribution of de Novo Fatty Acid Synthesis to Hepatic Steatosis and Insulin Resistance: Lessons from Genetically Engineered Mice. J. Clin. Invest. 118, 829–838 (2008).
9. Day, C. P. Clinical spectrum and therapy of non-alcoholic steatohepatitis. Dig. Dis. 30 Suppl 1, 69–73 (2012).
10. Lima-Cabello, E. et al. Enhanced expression of pro-inflammatory mediators and liver X-receptor-regulated lipogenic genes in non-alcoholic fatty liver disease and hepatitis C. Clin. Sci. (Lond). 120, 239–250 (2011).
11. Nakamuta, M. et al. Impact of cholesterol metabolism and the LXRalpha-SREBP-1c pathway on nonalcoholic fatty liver disease. Int. J. Mol. Med. 23, 603–608 (2009).
12. Kohjima, M. et al. Re-evaluation of fatty acid metabolism-related gene expression in nonalcoholic fatty liver disease. Int. J. Mol. Med. 20, 351–358 (2007).
13. Osei-Hyiaman, D. et al. Endocannabinoid Activation at Hepatic CB1 Receptors Stimulates Fatty Acid Synthesis and Contributes to Diet-Induced Obesity. J. Clin. Invest. 115, 1298–1305 (2005).
14. Jeong, W. et al. Paracrine activation of hepatic CB1 receptors by stellate cell-derived endocannabinoids mediates alcoholic fatty liver. Cell Metab. 7, 227–35 (2008).
15. Di Marzo, V., Piscitelli, F. & Mechoulam, R. Cannabinoids and Endocannabinoids in Metabolic Disorders with Focus on Diabetes. Handb. Exp. Pharmacol. 2011, 75–104 (2011).
16. Pacher, P. & Mechoulam, R. Is Lipid Signaling through Cannabinoid 2 Receptors Part of a Protective System? Prog. Lipid Res. 50, 193–211 (2011).
17. Pacher, P., Bátkai, S. & Kunos, G. The Endocannabinoid System as an Emerging Target of Pharmacotherapy. Pharmacol. Rev. 58, 389–462 (2006).
18. Mallat, A., Teixeira-Clerc, F., Deveaux, V., Manin, S. & Lotersztajn, S. The Endocannabinoid System as a Key Mediator During Liver Diseases: New Insights and Therapeutic Openings. Br. J. Pharmacol. In press, (2011).
19. Tam, J. et al. Peripheral CB1 cannabinoid receptor blockade improves cardiometabolic risk in mouse models of obesity. J. Clin. Invest. 120, 2953–2966 (2010).
20. Osei-Hyiaman, D. et al. Hepatic CB1 receptor is required for development of diet-induced steatosis, dyslipidemia, and insulin and leptin resistance in mice. J. Clin. Invest. 118, 3160–3169 (2008).
21. Gary-Bobo, M. et al. Rimonabant reduces obesity-associated hepatic steatosis and features of metabolic syndrome in obese Zucker fa/fa rats. Hepatology 46, 122–129 (2007).
22. Jourdan, T. et al. Antagonism of peripheral hepatic cannabinoid receptor-1 improves liver lipid metabolism in mice: evidence from cultured explants. Hepatology 55, 790–9 (2012).
23. Jourdan, T. et al. CB1 Antagonism Exerts Specific Molecular Effects on Visceral and Subcutaneous Fat and Reverses Liver Steatosis in Diet-Induced Obese Mice. Diabetes 59, 926–934 (2010).
24. Mallat, A., Teixeira-Clerc, F. & Lotersztajn, S. Cannabinoid signaling and liver therapeutics. J. Hepatol. (2013). doi:10.1016/j.jhep.2013.03.032; 10.1016/j.jhep.2013.03.032
25. Mendez-Sanchez, N. et al. Endocannabinoid Receptor CB2 in Nonalcoholic Fatty Liver Disease. Liver Int. 27, 215–219 (2007).

 

Acknowledgements:

This study was supported by the Ministerio de Ciencia e Innovación of the government of Spain (Grant no. SAF 2008-02278, to Cristóbal Richart), the Fondo de Investigación
Sanitaria (Grant no. PS09/01778, to Teresa Auguet), funds from Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR 2009 SGR 959 to Cristóbal Richart), Grup de Recerca en Medicina Aplicada URV (2010PFR-URV-B2-14 to Cristóbal Richart), and the Fundación Biociencia.

 

Contact:

Prof. Cristóbal Richart
Department of Internal Medicine
Hospital Universitari de Tarragona Joan XXIII
Universitat Rovira i Virgili
Mallafré Guasch, 4
43007 Tarragona
Catalonia, Spain
email: crichart.hj23.ics@gencat.cat

Figure-1
Figure 1. Mechanisms of CB1 involved in hepatic lipid accumulation. The activation of CB1 receptors in adipose tissue promotes LPL activity, which results in increased FFAs release into the liver. The activation of hepatic CB1 receptors contributes to liver fat accumulation by increased de novo hepatic lipogenesis, decreased fatty acid oxidation, and decreased secretion of TG-rich VLDL. LPL: lipoprotein lipase; FFAs: free fatty acids; TGs: triglycerides

 

Figure-2Figure 2. Liver CB1 and CB2 mRNA expression in morbidly obese women classified according to the liver pathology. NL: with normal liver histology; SS: simple steatosis; NASH: non-alcoholic steatohepatitis. Results are shown as mean ± SD.

 

Figure-3Table 1. Correlations between the expression of CB1 and CB2 and genes related to fatty acid synthesis, to fatty acid oxidation, uptake and transport, inflammation and adipokines in liver from morbidly obese women.

 

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